Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles

Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

The data generated in this study which are shown in scatter plots and line plots in main and supplementary figures are provided in the Source Data file.The raw data generated in this study for measurement of the Rac and Rho activity dynamics in migrating cells shown in Figure 4a-d and Supplementary Figure 4a-d are provided in the Github repositories along with analysis code (insert Github link).All other datasets generated during and/or analyzed during this study are available from the corresponding author on request.

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N/A N/A N/A Statistical methods to predetermine sample size were not implemented.For each condition, a sample size was chosen that represents an appropriate balance between technical resources and data variability.For high resolution microscopy, more than 30 cells were typically investigated.For low resolution imaging (e.g.tracking nuclei for cell migration measurements) a larger number of cells (typically about 500) were analysed.
For cell migration measurements via labeled nuclei, all cells that were detected for the whole time series were included in analyses.For high resolution imaging of transiently transfected cells, only those cells that express all constructs at adequate levels for quantitative analysis were included, as it is commonly done in the field.Furthermore, analysis via the ADAPT plugin was only possible if cells were completely imaged in the field of view and did not interact with other cells.
All reported results were replicated and reproduced at least 3 times.
All reported quantitative results are based on unbiased automated analysis and did not include subjective evaluations.Therefore, randomization of experimental conditions was not required.
All reported quantitative results are based on unbiased automated analysis and did not include subjective evaluations.Therefore, blinding of experimental conditions was not required.The cell lines were not tested regularly for mycoplasma contamination.
According to the ICLAC register, no commonly misidentified cell lines were used in this study.
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